Crystal Structure of the RUN Domain of the RAP2-interacting Protein x

Kukimoto-Niino, Mutsuko; Takagi, Tetsuo; Akasaka, Ryogo; Murayama, Kazutaka; Uchikubo‐Kamo, Tomomi; Terada, Takaho; Inoue, Makoto; Watanabe, Satoru; Tanaka, Akiko; Hayashizaki, Yoshihide; Kigawa, T.; Shirouzu, Mikako; Yokoyama, Shigeyuki

doi:10.1074/jbc.m604960200

Cited by 35 publications

(35 citation statements)

References 58 publications

(54 reference statements)

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“…The RUN domain was originally found in a group of proteins that interact with small GTPases (15)(16)(17). It was therefore proposed as a binding surface for small GTPases.…”

Section: Discussionmentioning

confidence: 99%

“…Rubicon is involved in autophagosome maturation and is likely involved in endosome maturation also (9,10). Rubicon contains 972 amino acids, with a coiled-coil domain, an FYVE-like domain, and a recognizable RUN domain that is shared by a group of proteins interacting with small GTPases (15)(16)(17).…”

Section: Composition Of the Rubicon-containing Pi3kc3 Complex-mentioning

confidence: 99%

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The RUN Domain of Rubicon Is Important for hVps34 Binding, Lipid Kinase Inhibition, and Autophagy Suppression

Sun

Zhang

Fan

et al. 2011

Journal of Biological Chemistry

127

111

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The class III phosphatidylinositol 3-kinase (PI3KC3) plays a central role in autophagy. Rubicon, a RUN domain-containing protein, is newly identified as a PI3KC3 subunit through its association with Beclin 1. Rubicon serves as a negative regulator of PI3KC3 and autophagosome maturation. The molecular mechanism underlying the PI3KC3 and autophagy inhibition by Rubicon is largely unknown. Here, we demonstrate that Rubicon interacts with the PI3KC3 catalytic subunit hVps34 via its RUN domain. The RUN domain contributes to the efficient inhibition of PI3KC3 lipid kinase activity by Rubicon. Furthermore, a Rubicon RUN domain deletion mutant fails to complement the autophagy deficiency in Rubicon-depleted cells. Hence, these results reveal a critical role of the Rubicon RUN domain in PI3KC3 and autophagy regulation. The class III PI3K (PI3KC3)2 is essential for autophagy, protein sorting, phagosome maturation, and cytokinesis (1-4). PI3KC3 is composed of the human Vps34 (hVps34) catalytic subunit and two regulatory subunits (p150 and Beclin 1) that are highly conserved from yeast to human. Functional equivalents of Beclin 1/hVps34/p150 in yeast (Atg6/Vps15/ Vps34) are essential for autophagy and vacuolar protein sorting (3, 5). The specificity of PI3KC3 in yeast is determined by different complex compositions. Two regulatory proteins, Atg14 and Vps38, direct the core PI3K complex to either the pre-autophagosome structure for autophagy or the endosome for vacuolar protein sorting (3, 6), respectively.Along with several other groups, we have recently purified a mammalian PI3KC3 holocomplex that includes hVps34, p150, Beclin 1, UVRAG, Barkor/Atg14(L), and Rubicon (also called p120 and Baron) (7-12). PI3KC3 forms two mutually exclusive protein subcomplexes that localize to different subcellular organelles and execute distinct functions. One complex is composed of the PI3KC3 core complex (hVps34, p150, and Beclin 1) and Barkor/Atg14(L). Barkor/Atg14(L) is the targeting factor for this complex to nascent autophagosomes (7). The other complex consists of the PI3KC3 core complex and UVRAG. UVRAG positively regulates PI3KC3 activity and autophagy maturation (13). UVRAG is also required for endocytosis through its interaction with C-VPS/HOPS (13,14). Rubicon (RUN domain protein as Beclin 1-interacting and cysteine-rich containing) is a newly identified PI3KC3 subunit (7-12). It serves as a negative regulator of autophagosome and endosome maturation (9,10,12).Understanding the functional mode and structure basis of Rubicon in autophagy is crucial. Here, we demonstrate that Rubicon resides in the UVRAG subcomplex of PI3KC3. Interestingly, we detected no direct interaction between Rubicon and Beclin 1, although it was originally purified in the Beclin 1 complex. Instead, Rubicon physically interacted with UVRAG and hVps34. We have mapped the interaction domain of Rubicon with hVps34 to the RUN domain. The RUN domain is critical for the function of Rubicon in suppressing PI3KC3 lipid kinase activity and autophagy maturation. EXPERI...

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“…The RUN domain was originally found in a group of proteins that interact with small GTPases (15)(16)(17). It was therefore proposed as a binding surface for small GTPases.…”

Section: Discussionmentioning

confidence: 99%

Section: Composition Of the Rubicon-containing Pi3kc3 Complex-mentioning

confidence: 99%

The RUN Domain of Rubicon Is Important for hVps34 Binding, Lipid Kinase Inhibition, and Autophagy Suppression

Sun

Zhang

Fan

et al. 2011

Journal of Biological Chemistry

127

111

View full text Add to dashboard Cite

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“…S1B). Moreover, the minimal Rap2-binding site of RUFY3 has been shown to be 40 amino acids larger than the predicted RUN domain (Kukimoto-Niino et al, 2006), indicating that the RUN domain alone is insufficient to recognize Rap2. This observation is very similar to our own results showing that the minimal Rab35-binding site of RUSC2 is larger than the predicted RUN domain (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…This is still an open question, because our findings clearly rule out the possibility that Rab is a common ligand of all RUN domains. Since some RUN domains have been shown to bind Rap, another type of small GTPase (Janoueix- Lerosey et al, 1998;Kukimoto-Niino et al, 2006;Yang et al, 2007), Rap may be a common ligand of RUN domains. However, we think that this possibility is also unlikely because of the very low sequence conservation among the known RUN domains (only one Tyr residue is invariant in all RUN domains; asterisk in Supplemental Fig.…”

Section: Discussionmentioning

confidence: 99%

Genome-wide Investigation of the Rab Binding Activity of RUN Domains: Development of a Novel Tool that Specifically Traps GTP-Rab35

Fukuda

Kobayashi

Ishibashi

et al. 2011

Cell Struct. Funct.

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ABSTRACT. 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 domaincontaining 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.

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“…1C). Recently, the crystal structure of its RUN domain was reported (30). We designated it singar1, together with Dr. Osamu Ohara (Kazusa DNA Research Institute), who initially reported its full cDNA sequence as KIAA0871 (AB020678) and Dr. Takahiro Nagase (Kazusa DNA Research Institute).…”

Section: Identification Of Singar1 By a Proteome Screening Of Stage 2mentioning

confidence: 99%

Singar1, a Novel RUN Domain-containing Protein, Suppresses Formation of Surplus Axons for Neuronal Polarity

Mori

Wada

Suzuki

et al. 2007

Journal of Biological Chemistry

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Although neuronal functions depend on their robust polarity, the mechanisms that ensure generation and maintenance of only a single axon remain poorly understood. Using highly sensitive two-dimensional electrophoresis-based proteomics, we identified here a novel protein, single axon-related (singar)1/KIAA0871/RPIPx/RUFY3, which contains a RUN domain and is predominantly expressed in the brain. Singar1 expression became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Singar1 was diffusely localized in hippocampal neurons and moderately accumulated in growth cones of minor processes and axons. Overexpression of singar1 did not affect normal neuronal polarization but suppressed the formation of surplus axons induced by excess levels of shootin1, a recently identified protein located upstream of phosphoinositide-3-kinase and involved in neuronal polarization. Conversely, reduction of the expression of singar1 and its splicing variant singar2 by RNA interference led to an increase in the population of neurons bearing surplus axons, in a phosphoinositide-3-kinase-dependent manner. Overexpression of singar2 did not suppress the formation of surplus axons induced by shootin1. We propose that singar1 ensures the robustness of neuronal polarity by suppressing formation of surplus axons.Most neurons develop polarity by forming a single long axon and multiple short dendrites (1, 2). The ability of neurons to develop and maintain polarity is essential for their basic functions. The polarization processes have been extensively studied in cultured hippocampal neurons (1, 3). These cells first form several minor processes, any of which appears capable of becoming either an axon or a dendrite, during the first 12-24 h after plating (stage 2). One of the neurites then rapidly elongates to acquire axonal characteristics (stage 3), whereas the others later become dendrites (stage 4). Hippocampal neurons must use a robust internal mechanism that ensures polarization as they regenerate a single axon and multiple dendrites even when polarity is altered by axonal amputation (4, 5).Recent studies have begun to reveal molecules involved in neuronal polarization (6 -8). Localization, overexpression, and loss of function studies showed that spatially localized intracellular signals of a number of molecules, such as phosphoinositide-3-kinase (PI 3-kinase) 2 (9), phosphatidylinositol (3, 4, 5) triphosphate (10), Akt (11), glycogen synthase kinase-3␤ (11-13), collapsin response mediator protein-2 (CRMP-2) (12, 14), mPar3/mPar6/atypical protein kinase C complex (9, 15), Cdc42 (15, 16), Rap1B (16), STEF/Tiam1 (15, 17), Rac (15), adenomatous polyposis coli (18), JNK (19), and DOCK7 (20) are involved in axon specification for neuronal polarity formation. As downstream events, these pathways are thought to regulate cytoskeletal networks of actin filaments (21) and microtubules (20,22).In contrast to the progress in defining the mechanisms of axon specification, our knowledge of how neurons g...

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Crystal Structure of the RUN Domain of the RAP2-interacting Protein x

Cited by 35 publications

References 58 publications

The RUN Domain of Rubicon Is Important for hVps34 Binding, Lipid Kinase Inhibition, and Autophagy Suppression

The RUN Domain of Rubicon Is Important for hVps34 Binding, Lipid Kinase Inhibition, and Autophagy Suppression

Genome-wide Investigation of the Rab Binding Activity of RUN Domains: Development of a Novel Tool that Specifically Traps GTP-Rab35

Singar1, a Novel RUN Domain-containing Protein, Suppresses Formation of Surplus Axons for Neuronal Polarity

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