Zhongzhen Nie

Arf and its many interactors

Arf GTP-binding proteins regulate membrane traffic and actin remodeling. Similar to other GTP-binding proteins, a complex of Arf-GTP with an effector protein mediates Arf function. Arf interacts with at least three qualitatively different types of effectors. First, it interacts with structural proteins, the vesicle coat proteins. The second type of effector is lipid-metabolizing enzymes, and the third comprises those proteins that bind to Arf-GTP but whose biochemical or biological functions are not yet clearly defined. Arf interacts with two other families of proteins, the exchange factors and the GTPase-activating proteins. Recent work examining the functional relationships among the diverse Arf interactors has led to reconsideration of the prevailing paradigms for Arf action.

Arf GAPs and membrane traffic

The selective transfer of material between membrane-delimited organelles is mediated by protein-coated vesicles. In many instances, formation of membrane trafficking intermediates is regulated by the GTP-binding protein Arf. Binding and hydrolysis of GTP by Arf was originally linked to the assembly and disassembly of vesicle coats. Arf GTPase-activating proteins (GAPs), a family of proteins that induce hydrolysis of GTP bound to Arf, were therefore proposed to regulate the disassembly and dissociation of vesicle coats. Following the molecular identification of Arf GAPs, the roles for GAPs and GTP hydrolysis have been directly examined. GAPs have been found to bind cargo and known coat proteins as well as directly contribute to vesicle formation, which is consistent with the idea that GAPs function as subunits of coat proteins rather than simply Arf inactivators. In addition, GTP hydrolysis induced by GAPs occurs largely before vesicle formation and is required for sorting. These results are the primary basis for modifications to the classical model for the function of Arf in transport vesicle formation, including a recent proposal that Arf has a proofreading, rather than a structural, role.

Molecular Aspects of the Cellular Activities of ADP-Ribosylation Factors

Adenosine diphosphate-ribosylation factor (Arf) proteins are members of the Arf arm of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins. Arfs are named for their activity as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of the heterotrimeric G protein Gs. Physiologically, Arfs regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. In each case, the controlled binding and hydrolysis of GTP is critical to Arf function. The activities of some guanine nucleotide exchange factors (GEFs) and guanosine triphosphatase (GTPase)-activating proteins (GAPs) are stimulated by phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA), likely providing both a means to respond to regulatory signals and a mechanism to coordinate GTP binding and hydrolysis. Arfs affect membrane traffic in part by recruiting coat proteins, including COPI and clathrin adaptor complexes, to membranes. However, Arf function likely involves many additional biochemical activities. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase with the consequent production of PA and PIP2, respectively. In addition to mediating Arfs effects on membrane traffic and the actin cytoskeleton, PA and PIP2 are involved in the regulation of Arf. Arf also works with Rho family proteins to affect the actin cytoskeleton. Several Arf-binding proteins suspected to be effectors have been identified in two-hybrid screens. Arf-dependent biochemical activities, actin cytoskeleton changes, and membrane trafficking may be integrally related. Understanding Arfs role in complex cellular functions such as protein secretion or cell movement will involve a description of the temporal and spatial coordination of these multiple Arf-dependent events. The adenosine diphosphate-ribosylation factor (Arf) proteins, first identified as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of Gs, are a family of guanosine triphosphate-binding proteins that regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. Arf activity is in part mediated by binding and recruitment of vesicle coat proteins. Arf proteins also bind to and activate phospholipase D and phosphatidylinositol 4-phosphate 5-kinase, producing phosphatidic acid and phosphatidylinositol 4,5-bisphosphate, respectively. These two lipids contribute to the effects of Arf on membrane traffic and the actin cytoskeleton. In addition, phosphatidic acid and phosphatidylinositol 4,5-bisphosphate are involved in the regulation of Arf, interacting with both guanine nucleotide exchange factors and guanosine triphosphatase-activating proteins. Additional Arf-binding proteins have been identified through two-hybrid screens. The molecular mechanisms by which Arf-binding proteins and phospholipids contribute to Arfs physiologic functions are being discovered. Abbreviations: Arf, ADP-ribosylation factor; BFA, brefeldin A; COP, coat protein complex; FA, focal adhesion; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate.

Consensus nomenclature for the human ArfGAP domain-containing proteins

At the FASEB summer research conference on “Arf Family GTPases”, held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.

A role for BARS at the fission step of COPI vesicle formation from Golgi membrane

The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular‐like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl‐coenzyme A (p‐coA) and ARFGAP1, a GTPase‐activating protein (GAP) for ADP‐Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin‐A ADP‐Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p‐coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p‐coA and GAP are linked in this process.

A BAR Domain in the N Terminus of the Arf GAP ASAP1 Affects Membrane Structure and Trafficking of Epidermal Growth Factor Receptor

BACKGROUNDnArf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1.nnnRESULTSnASAP1s N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect.nnnCONCLUSIONSnThe N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

Src-Dependent Phosphorylation of ASAP1 Regulates Podosomes

ABSTRACT Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

Specific Regulation of the Adaptor Protein Complex AP-3 by the Arf GAP AGAP1

Arf1 regulates membrane trafficking at several membrane sites by interacting with at least seven different vesicle coat proteins. Here, we test the hypothesis that Arf1-dependent coats are independently regulated by specific interaction with Arf GAPs. We find that the Arf GAP AGAP1 directly associates with and colocalizes with AP-3, a coat protein complex involved in trafficking in the endosomal-lysosomal system. Binding is mediated by the PH domain of AGAP1 and the delta and sigma3 subunits of AP-3. Overexpression of AGAP1 changes the cellular distribution of AP-3, and reduced expression of AGAP1 renders AP-3 resistant to brefeldin A. AGAP1 overexpression does not affect the distribution of other coat proteins, and AP-3 distribution is not affected by overexpression of other Arf GAPs. Cells overexpressing AGAP1 also exhibit increased LAMP1 trafficking via the plasma membrane. Taken together, these results support the hypothesis that AGAP1 directly and specifically regulates AP-3-dependent trafficking.

ARAP3 is transiently tyrosine phosphorylated in cells attaching to fibronectin and inhibits cell spreading in a RhoGAP-dependent manner

ARAP3 is a GTPase activating protein (GAP) for Rho and Arf GTPases that is implicated in phosphoinositide 3-kinase (PI 3-kinase) signalling pathways controlling lamellipodia formation and actin stress fibre assembly. We have identified ARAP3 as a phosphorylated target of protein tyrosine kinases. In cells, ARAP3 was tyrosine phosphorylated when co-expressed with Src-family kinases (SFKs), upon stimulation with growth factors and during adhesion to the extracellular matrix (ECM) substrate fibronectin. Adhesion-induced phosphorylation of ARAP3 was suppressed by selective inhibitors of Src-family kinases and PI 3-kinase and by a Src dominant interfering mutant. Inducible expression of ARAP3 in HEK293 epithelial cells resulted in increased cell rounding, membrane process formation and cell clustering on ECM substrates. In contrast, ARAP3 dramatically slowed the kinetics of cell spreading on fibronectin but had no effect on cell adhesion. These effects of ARAP3 required a functional Rho GAP domain and were associated with reduced cellular levels of active RhoA and Rac1 but did not require the sterile alpha motif (SAM) or Arf GAP domains. Mutation of two phosphorylation sites, Y1399 and Y1404, enhanced some ARAP3 activities, suggesting that ARAP3 may be negatively regulated by phosphorylation on these tyrosine residues. These results implicate ARAP3 in integrin-mediated tyrosine kinase signalling pathways controlling Rho GTPases and cell spreading.

ASAP3 Is a Focal Adhesion-associated Arf GAP That Functions in Cell Migration and Invasion

ASAP3, an Arf GTPase-activating protein previously called DDEFL1 and ACAP4, has been implicated in the pathogenesis of hepatocellular carcinoma. We have examined in vitro and in vivo functions of ASAP3 and compared it to the related Arf GAP ASAP1 that has also been implicated in oncogenesis. ASAP3 was biochemically similar to ASAP1: the pleckstrin homology domain affected function of the catalytic domain by more than 100-fold; catalysis was stimulated by phosphatidylinositol 4,5-bisphosphate; and Arf1, Arf5, and Arf6 were used as substrates in vitro. Like ASAP1, ASAP3 associated with focal adhesions and circular dorsal ruffles. Different than ASAP1, ASAP3 did not localize to invadopodia or podosomes. Cells, derived from a mammary carcinoma and from a glioblastoma, with reduced ASAP3 expression had fewer actin stress fiber, reduced levels of phosphomyosin, and migrated more slowly than control cells. Reducing ASAP3 expression also slowed invasion of mammary carcinoma cells. In contrast, reduction of ASAP1 expression had no effect on migration or invasion. We propose that ASAP3 functions nonredundantly with ASAP1 to control cell movement and may have a role in cancer cell invasion. In comparing ASAP1 and ASAP3, we also found that invadopodia are dispensable for the invasive behavior of cells derived from a mammary carcinoma.