DENN domain proteins: regulators of Rab GTPases

The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways.     

Molecular cloning,structural analysis,and expression of a human IRLB,MYC promoter-binding protein: new DENN domain-containing protein family emerges

IRLB was originally identified as a partial cDNA clone, encoding a 191-aa protein binding the interferon-stimulated response element (ISRE) in the P2 promoter of human MYC. Here, we cloned the full-size IRLB using different bioinformatics tools and an RT-PCR approach. The full-size gene encompasses 131 kb within chromosome 15q22 and consists of 32 exons. IRLB is transcribed as a 6.6-kb mRNA encoding a protein of 1865 aa. IRLB is ubiquitously expressed and its expression is regulated in a growth- and cell cycle-dependent manner. In addition to the ISRE-binding domain IRLB contains a tripartite DENN domain, a nuclear localization signal, two PPRs, and a calmodulin-binding domain. The presence of DENN domains predicts possible interactions of IRLB with GTPases from the Rab family or regulation of growth-induced MAPKs. Strongly homologous proteins were identified in all available vertebrate genomes as well as in Caenorhabditis elegans and Drosophila melanogaster. In human and mouse a family of IRLB proteins exists, consisting of at least three members.     

KIF1Bbeta- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD

Synaptic proteins are synthesized in the cell body and transported down the axon by microtubule-dependent motors. We previously reported that KIF1Bbeta and KIF1A motors are essential for transporting synaptic vesicle precursors; however the mechanisms that regulate transport, as well as cargo recognition and control of cargo loading and unloading remain largely unknown. Here, we show that DENN/MADD (Rab3-GEP) is an essential part of the regulation mechanism through direct interaction with the stalk domain of KIF1Bbeta and KIF1A. We also show that DENN/MADD binds preferentially to GTP-Rab3 and acts as a Rab3 effector. These molecular interactions are fundamental as sequential genetic perturbations revealed that KIF1Bbeta and KIF1A are essential for the transport of DENN/MADD and Rab3, whereas DENN/MADD is essential for the transport of Rab3. GTP-Rab3 was more effectively transported than GDP-Rab3, suggesting that the nucleotide state of Rab3 regulates axonal transport of Rab3-carrying vesicles through preferential interaction with DENN/MADD.     

Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors

Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction.     

Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors

A key requirement for Rab function in membrane trafficking is site-specific activation by GDP-GTP exchange factors (GEFs), but the majority of the 63 human Rabs have no known GEF. We have performed a systematic characterization of the 17 human DENN domain proteins and demonstrated that they are specific GEFs for 10 Rabs. DENND1A/1B localize to clathrin patches at the plasma membrane and activate Rab35 in an endocytic pathway trafficking Shiga toxin to the trans-Golgi network. DENND2 GEFs target to actin filaments and control Rab9-dependent trafficking of mannose-6-phosphate receptor to lysosomes. DENND4 GEFs target to a tubular membrane compartment adjacent to the Golgi, where they activate Rab10, which suggests a function in basolateral polarized sorting in epithelial cells that compliments the non-DENN GEF Sec2 acting on Rab8 in apical sorting. DENND1C, DENND3, DENND5A/5B, MTMR5/13, and MADD activate Rab13, Rab12, Rab39, Rab28, and Rab27A/27B, respectively. Together, these findings provide a basis for future studies on Rab regulation and function.     

Dual role of DENN/MADD (Rab3GEP) in neurotransmission and neuroprotection

DENN/MADD is a component of a signalling protein complex that is localized to the cytosol and exerts multiple functions by using different binding partners. Human DENN/MADD is physically the same death-domain protein as rat Rab3 GDP/GTP exchange protein (Rab3GEP). DENN/MADD regulates the recycling of Rab3 small G proteins under normal conditions and has an essential role in Ca(2+)-dependent neurotransmitter release and exocytosis. It is also involved in blocking the apoptosis of neuronal cells under conditions of cytotoxic stress. Recent research supports an important role for DENN/MADD in neuroprotection: reduced endogenous DENN/MADD expression and enhanced pro-apoptotic signalling has been found in brains affected by Alzheimer's disease.     

Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton

The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)-domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1-3/DENND1A-C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.     

Genome-wide investigation of the Rab binding activity of RUN domains: development of a novel tool that specifically traps GTP-Rab35

The RUN domain is a less conserved protein motif that consists of approximately 70 amino acids, and because RUN domains are often found in proteins involved in the regulation of Rab small GTPases, the RUN domain has been suggested to be involved in Rab-mediated membrane trafficking, possibly as a Rab-binding site. However, since the Rab binding activity of most RUN domains has never been investigated, in this study we performed a genome-wide analysis of the Rab binding activity of the RUN domains of 19 different RUN domain-containing proteins by yeast two-hybrid assays with 60 different Rabs as bait. The results showed that only six of them interact with specific Rab isoforms with different Rab binding specificity, i.e., DENND5A/B with Rab6A/B, PLEKHM2 with Rab1A, RUFY2/3 with Rab33, and RUSC2 with Rab1/Rab35/Rab41. We also identified the minimal functional Rab35-binding site of RUSC2 (amino acid residues 982-1199) and succeeded in developing a novel GTP-Rab35-specific trapper, which we named RBD35 (Rab-binding domain specific for Rab35). Recombinant RBD35 was found to trap GTP-Rab35 specifically both in vitro and in PC12 cells, and overexpression of fluorescently tagged RBD35 in PC12 cells strongly inhibited nerve growth factor-dependent neurite outgrowth.     

DENN domain-containing protein FAM45A regulates the homeostasis of late/multivesicular endosomes

DENN (differentially expressed in normal cells and neoplasia) domain-containing proteins are a family of guanine nucleotide exchange factors (GEFs) for Rab small GTPases and coordinate a plethora of intracellular membrane trafficking events. FAM45A is a non-classical DENN domain protein, whose function was unknown. In this study, we characterized cellular roles of FAM45A. We found that FAM45A localized mainly in late/multivesicular endosomes. Depletion of FAM45A resulted in clustering of endosomes to the perinuclear region. The endocytosis of EGF receptor was impaired in FAM45A knockdown cells due to a delay in the early-to-late endosome transition. Furthermore, the secretion of selected exosome subpopulations was also attenuated in FAM45A knockdown cells. Consistent with these results, Rab27a and Rab27b, two Rabs involved in endosome motility and exosome biogenesis, were found to act downstream of FAM45A pathway. FAM45A colocalized with Rab27a/b and formed complex with them in a nucleotide-dependent manner. Taken together, FAM45A defines a novel regulatory step in the homeostasis of late endocytic pathway, including endosomal positioning, maturation and secretion, possibly through activating Rab proteins such as Rab27a/b.     

Mapping the interactions between a RUN domain from DENND5/Rab6IP1 and sorting nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.     

Evolutionarily conserved structural and functional roles of the FYVE domain

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12-13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4'. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.     

Synaptotagmin-like protein 5: a novel Rab27A effector with C-terminal tandem C2 domains

Synaptotagmin-like proteins 1-4 (Slp1-4) are new members of the carboxyl-terminal-type (C-type) tandem C2 proteins and are classified as a subfamily distinct from the synaptotagmin and the Doc2 families, because the Slp family contains a unique homology domain at the amino terminus, referred to as the Slp homology domain (SHD). We previously showed that the SHD functions as a binding site for Rab27A, which is associated with human hemophagocytic syndrome (Griscelli syndrome) [J. Biol. Chem. 277 (2002) 9212; J. Biol. Chem. 277 (2002) 12432]. In the present study, we identified a novel member of the Slp family, Slp5. The same as other Slp family members, the SHD of Slp5 preferentially interacted with the GTP-bound form of Rab27A and marginally with Rab3A and Rab6A, both in vitro and in intact cells, but not with other Rabs tested (Rab1, Rab2, Rab4A, Rab5A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab28, and Rab37). However, unlike other members of the Slp family, expression of Slp5 mRNA was highly restricted to human placenta and liver. Expression of Slp5 protein and in vivo association of Slp5 with Rab27A in the mouse liver were further confirmed by immunoprecipitation. The results suggest that Slp5 might be involved in Rab27A-dependent membrane trafficking in specific tissues.     

Tyr-phosphorylation signals translocate RIN3, the small GTPase Rab5-GEF, to early endocytic vesicles

The small GTPase Rab5 plays a key role in early endocytic pathway, and its activation requires guanine-nucleotide exchange factors (GEFs). Rab5-GEFs share a conserved VPS9 domain for the GEF action, and RIN3 containing additional domains, such as Src-homology 2, RIN-family homology (RH), and Ras-association (RA), was identified as a new Rab5-GEF. However, precise functions of the additional domains and the activation mechanism of RIN3 remain unknown. Here, we found tyrosine-phosphorylation signals are involved in the Rab5-GEF activation. Treatment of HeLa cells with pervanadate translocates RIN3 from cytoplasm to the Rab5-positive vesicles. This RIN3 translocation was applied to various mutants lacking each domain of RIN3. Our present results suggest that a Ras GTPase(s) activated by tyrosine-phosphorylation signals interacts with the inhibitory RA domain, resulting in an active conformation of RIN3 as a Rab5-GEF and that RIN-unique RH domain constitutes a Rab5-binding region for the progress of GEF action.     

Characterization of a Listeria monocytogenes protein interfering with Rab5a

Listeria monocytogenes (LM) phagocytic strategy implies recruitment and inhibition of Rab5a. Here, we identify a Listeria protein that binds to Rab5a and is responsible for Rab5a recruitment to phagosomes and impairment of the GDP/GTP exchange activity. This protein was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Listeria (p40 protein, Lmo 2459). The p40 protein was found within the phagosomal membrane. Analysis of the sequence of LM p40 protein revealed two enzymatic domains: the nicotinamide adenine dinucleotide (NAD)-binding domain at the N-terminal and the C-terminal glycolytic domain. The putative ADP-ribosylating ability of this Listeria protein located in the N-terminal domain was examined and showed some similarities to the activity and Rab5a inhibition exerted by Pseudomonas aeruginosa ExoS onto endosome–endosome fusion. Listeria p40 caused Rab5a-specific ADP ribosylation and blocked Rab5a-exchange factor (Vps9) and GDI interaction and function, explaining the inhibition observed in Rab5a-mediated phagosome–endosome fusion. Meanwhile, ExoS impaired Rab5-early endosomal antigen 1 (EEA1) interaction and showed a wider Rab specificity. Listeria GAPDH might be the first intracellular gram-positive enzyme targeted to Rab proteins with ADP-ribosylating ability and a putative novel virulence factor.     

Folliculin directs the formation of a Rab34–RILP complex to control the nutrient‐dependent dynamic distribution of lysosomes

The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt–Hoge–Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri‐nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi‐associated small GTPase Rab34. Rab34‐positive peri‐nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34‐induced peri‐nuclear lysosome clustering. FLCN interacts directly via its C‐terminal DENN domain with the Rab34 effector RILP. Using purified recombinant proteins, we show that the FLCN‐DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP. We propose a model whereby starvation‐induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34‐positive peri‐nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell.     

Subunit organization and Rab interactions of Vps-C protein complexes that control endolysosomal membrane traffic

Traffic through late endolysosomal compartments is regulated by sequential signaling of small G proteins of the Rab5 and Rab7 families. The Saccharomyces cerevisiae Vps-C protein complexes CORVET (class C core vacuole/endosome tethering complex) and HOPS (homotypic fusion and protein transport) interact with endolysosomal Rabs to coordinate their signaling activities. To better understand these large and intricate complexes, we performed interaction surveys to assemble domain-level interaction topologies for the eight Vps-C subunits. We identified numerous intersubunit interactions and up to six Rab-binding sites. Functional modules coordinate the major Rab interactions within CORVET and HOPS. The CORVET-specific subunits, Vps3 and Vps8, form a subcomplex and physically and genetically interact with the Rab5 orthologue Vps21. The HOPS-specific subunits, Vps39 and Vps41, also form a subcomplex. Both subunits bind the Rab7 orthologue Ypt7, but with distinct nucleotide specificities. The in vivo functions of four RING-like domains within Vps-C subunits were analyzed and shown to have distinct functions in endolysosomal transport. Finally, we show that the CORVET- and HOPS-specific subunits Vps3 and Vps39 bind the Vps-C core through a common region within the Vps11 C-terminal domain (CTD). Biochemical and genetic experiments demonstrate the importance of these regions, revealing the Vps11 CTD as a key integrator of Vps-C complex assembly, Rab signaling, and endosomal and lysosomal traffic.     

D-AKAP2 Interacts with Rab4 and Rab11 through Its RGS Domains and Regulates Transferrin Receptor Recycling

Dual-specific A-kinase-anchoring protein 2 (D-AKAP2/AKAP10), which interacts at its carboxyl terminus with protein kinase A and PDZ domain proteins, contains two tandem regulator of G-protein signaling (RGS) domains for which the binding partners have remained unknown. We show here that these RGS domains interact with Rab11 and GTP-bound Rab4, the first demonstration of RGS domains binding small GTPases. Rab4 and Rab11 help regulate membrane trafficking through the endocytic recycling pathways by recruiting effector proteins to specific membrane domains. Although D-AKAP2 is primarily cytosolic in HeLa cells, a fraction of the protein localizes to endosomes and can be recruited there to a greater extent by overexpression of Rab4 or Rab11. D-AKAP2 also regulates the morphology of the Rab11-containing compartment, with co-expression causing accumulation of both proteins on enlarged endosomes. Knockdown of D-AKAP2 by RNA interference caused a redistribution of both Rab11 and the constitutively recycling transferrin receptor to the periphery of cells. Knockdown also caused an increase in the rate of transferrin recycling, suggesting that D-AKAP2 promotes accumulation of recycling proteins in the Rab4/Rab11-positive endocytic recycling compartment.     

Insulin-stimulated Interaction between insulin receptor substrate 1 and p85alpha and activation of protein kinase B/Akt require Rab5

Binding of insulin to the insulin receptor initiates a cascade of protein phosphorylation and effector recruitment events leading to the activation of multiple distinct signaling pathways. Previous studies suggested that the diversity and specificity of insulin signal transduction are accomplished by both subcellular localization of receptor and the selective activation of downstream signaling molecules. The small GTPase Rab5 is a key regulator of endocytosis. Three Rab5 isoforms (Rab5a, -5b, and -5c) have been identified. Here we exploited the RNA interference technique to specifically knock down individual Rab5 isoforms to determine the cellular function of Rab5 in distinct insulin signaling pathways. Small interference RNA against a single Rab5 isoform had no effect on protein kinase B (PKB)/Akt or MAPK activation by insulin in NIH3T3 cells overexpressing human insulin receptor. However, simultaneous knockdown of all three Rab5 isoforms dramatically attenuated PKB/Akt activation by insulin without affecting MAPK activation. This inhibition of PKB/Akt activation was because of the impaired interaction between insulin receptor substrate 1 and the p85alpha subunit of phosphatidylinositol 3-kinase. These results indicate a requirement of Rab5 in presenting p85 to insulin receptor substrate 1. Additional evidence supporting a role for Rab5 was suggested by studies with GAPex-5, a vps9 domain containing exchange factor. Down-regulation of GAPex-5 impaired insulin-stimulated PKB/Akt activation. Collectively, this study indicates the involvement of Rab5 in insulin signaling.     

C9orf72: At the intersection of lysosome cell biology and neurodegenerative disease

The discovery that expansion of a hexanucleotide repeat within a noncoding region of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia raised questions about C9orf72 protein function and potential disease relevance. The major predicted structural feature of the C9orf72 protein is a DENN (differentially expressed in normal and neoplastic cells) domain. As DENN domains are best characterized for regulation of specific Rab GTPases, it has been proposed that C9orf72 may also act through regulation of a GTPase target. Recent genetic and cell biological studies furthermore indicate that the C9orf72 protein functions at lysosomes as part of a larger complex that also contains the Smith‐Magenis chromosome region 8 (SMCR8) and WD repeat‐containing protein 41 (WDR41) proteins. An important role for C9orf72 at lysosomes is supported by defects in lysosome morphology and mTOR complex 1 (mTORC1) signaling arising from C9orf72 KO in diverse model systems. Collectively, these new findings define a C9orf72‐containing protein complex and a lysosomal site of action as central to C9orf72 function and provide a foundation for the elucidation of direct physiological targets for C9orf72. Further elucidation of mechanisms whereby C9orf72 regulates lysosome function will help to determine how the reductions in C9orf72 expression levels that accompany hexanucleotide repeat expansions contribute to disease pathology.