RINL, Guanine Nucleotide Exchange Factor Rab5-Subfamily, Is Involved in the EphA8-Degradation Pathway with Odin

The Rab family of small guanosine triphosphatases (GTPases) plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs). Ras and Rab interactor (or Ras interaction/interference)-like (RINL), which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM) domain-containing (Anks) protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin

The CDC42-Interacting Protein 4 Controls Epithelial Cell Cohesion and Tumor Dissemination

SummaryThe role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-β1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors

RhoA activation participates in rearrangement of processing bodies and release of nucleated AU-rich mRNAs

Cytoplasmic ribonucleoprotein granules, known as processing bodies (P-bodies), contain a common set of conserved RNA-processing enzymes, and mRNAs with AU-rich elements (AREs) are delivered to P-bodies for translational silencing. Although the dynamics of P-bodies is physically linked to cytoskeletal network, it is unclear how small GTPases are involved in the P-body regulation and the ARE-mRNA metabolism. We found here that glucose depletion activates RhoA GTPase and alters the P-body dynamics in HeLa cells. These glucose-depleted effects are reproduced by the overexpression of the RhoA-subfamily GTPases and conversely abolished by the inhibition of RhoA activation. Interestingly, both RhoA activation and glucose depletion inhibit the mRNA accumulation and degradation. These findings indicate that RhoA participates in the stress-induced rearrangement of P-bodies and the release of nucleated ARE-mRNAs for their stabilization

GEF activity of RINL for Rab5 subfamily proteins.

<p>(A–D) HEK293T cells expressing myc-Rab5b (A), Rab21 (B), Rab22 (C), or Rab31 (D) and FLAG-mock, RINL, RIN3, or Rabex-5 were metabolically radiolabeled with ³²P_i for 4 hours. Myc-Rab5 subfamily proteins were immunoprecipitated with an anti-myc monoclonal antibody, and nucleotides associating with each Rab protein were separated by thin-layer chromatography. The radioactivity of GTP and GDP was quantified, and the percentages (%) of each GTP-bound Rab are shown. Total lysates (bottom) and immunoprecipitated samples (middle) from the radiolabeled cells were separated by SDS-PAGE and immunoblotted with anti-FLAG and anti-myc antibodies, respectively. *p<0.05 vs. mock-transfected cells. (E) Myc-Rab3a, 7a, or 11a was co-transfected with FLAG-mock or RINL into HEK293T cells. The percentages of each GTP-bound Rab member in the metabolically radiolabeled cells are shown as described in (A). (F) Myc-Rab5b was co-transfected with wild type (WT), or the DP_AA or YT_AA mutant of FLAG-RINL into HEK293T cells. The percentages of GTP-Rab5b in the metabolically radiolabeled cells are shown as described in (A). Total lysates (bottom) and immunoprecipitated samples (middle) from the radiolabeled cells were separated by SDS-PAGE and immunoblotted with anti-FLAG and anti-myc antibodies, respectively. All data were obtained from more than three independent experiments and are shown as the mean ± S.E. (error bars). **p<0.01 vs. mock-transfected cells.</p

Identification of odin/Anks1a as an interacting molecule with RINL.

<p>(A) HeLa cell lysates were immunoprecipitated with normal rat IgG or anti-odin antibody, followed by immunoblotting with antibodies as indicated. (B) FLAG-RIN family or FLAG-mock were transfected into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with antibodies as indicated. (C) FLAG-RINL and the indicated deletion mutants of myc-odin were transiently transfected into HEK293T cells. Cells lysates were immunoprecipitated with anti-myc antibody, followed by immunoblotting with antibodies as shown. (D) The indicated deletion mutants of FLAG-RINL were transiently transfected into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with antibodies as indicated. (E) Myc-odin and V5-RINL were co-transfected with FLAG-tagged constitutively active (CA, lanes 2 and 4) or mock (lanes 1 and 3) into HEK293T cells. Cell lysates were immunoprecipitated with anti-myc antibody, followed by immunoblotting with antibodies as indicated. Aliquots of total lysates were also immunoblotted with antibodies as indicated.</p

Ski7p G protein interacts with the exosome and the Ski complex for 3′-to-5′ mRNA decay in yeast

Two cytoplasmic mRNA-decay pathways have been characterized in yeast, and both are initiated by shortening of the 3′-poly(A) tail. In the major 5′-to-3′ decay pathway, the deadenylation triggers removal of the 5′-cap, exposing the transcript body for 5′-to-3′ degradation. An alternative 3′-to-5′ decay pathway also follows the deadenylation and requires two multi-complexes: the exosome containing various 3′-exonucleases and the Ski complex consisting of the RNA helicase Ski2p, Ski3p and Ski8p. In addition, Ski7p, which has an N-terminal domain and a C-terminal elongation factor 1α-like GTP-binding domain, is involved in the 3′-to-5′ decay. However, physical interaction between the exosome and the Ski complex, together with the function of Ski7p, has remained unknown. Here we report that the N domain of Ski7p is required and sufficient for the 3′-to-5′ decay. Furthermore, the exosome and the Ski complex interact with the different regions of Ski7p N domain, and both interactions are required for the 3′-to-5′ decay. Thus, Ski7p G protein appears to function as a signal-coupling factor between the two multi-complexes operating in the 3′-to-5′ mRNA-decay pathway

RINL forms a ternary complex with odin and EphA8, and RINL affects the degradation of the EphA8 receptor.

<p>(A) HEK293T cells were co-transfected with EphA8-FLAG, HaloTag-odin, and myc-RINL (+) or mock (−) plasmids as indicated, and cell lysates were immunoprecipitated with anti-myc antibody. Immunoprecipitated fractions and total lysates were immunoblotted with antibodies as indicated. (B and C) HeLa cells were transfected with EphA8-FLAG and myc-RINL or mock plasmids, and total lysates were immunoblotted with antibodies as indicated. ΔSH2; SH2 domain-deleted mutant. The data obtained from three independent experiments are shown (C) as the mean ± S.E. (error bars). *, p<0.05 vs. mock-transfected cells. N.S., not significant. (D and E) HEK293T cells were transfected with EphA8-FLAG and myc-RINL or mock plasmids, and total lysates were immunoblotted with antibodies as indicated. WT; wild type. The data obtained from three independent experiments are shown (E) as the mean ± S.E. (error bars). *, p<0.05 vs. mock-transfected cells. (F and G) HeLa cells were transfected with 30 pmol scrambled negative control (NC) or RINL-specific siRNA. 24 hours after the transfection, these cells were transfected with EphA8-FLAG and siRNA-resistant FLAG-RINL, and incubated for 48 hours. Total proteins from the cell lysates were subjected to SDS-PAGE and immunoblotted (IB) with antibodies as indicated. The data obtained from three independent experiments are shown (G) as the mean ± S.E. (error bars). **, p<0.01 vs. NC-transfected cells. *, p<0.05 vs. siRNA-transfected cells with FLAG-mock plasmid transfection. (H and I) HeLa cells were transfected with EphA8-FLAG and FLAG-RINL (+, lanes 2–5) or mock plasmids (−, lane 1), and total lysates were immunoblotted with antibodies as indicated. These cells were non-treated (NT, lanes 1 and 2), or treated with MG132 (20 µM, lane 3), leupeptin (100 µg/ml, lane 4), or bafilomycin (200 nM, lane 5) for 3 hours. Total lysates were immunoblotted with antibodies as indicated. The data obtained from three independent experiments are shown (I) as the mean ± S.E. (error bars). **, p<0.01 vs. mock-transfected cells. *, p<0.05 vs. non-treatment cells transfected with RINL.</p

Structure of RINL.

<p>(A) Diagram of the structural features of RIN family members. The lower numbers represent the amino acid residues. (B) FLAG-RIN1, RIN2, RIN3, and RINL were transiently co-transfected with myc-amphiphysin II (amph II) into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-myc and anti-FLAG antibodies. Total lysates were immunoblotted with anti-myc antibody. (C) Cell lysates from HEK293T cells were applied to a Superdex 200 Prep Grade gel filtration column. The elution position was compared with those of the globular size markers (upper panel). The fractions (0.5 ml) eluted from the column and total lysate (tot.) were analyzed by SDS-PAGE, and proteins were immunoblotted with anti-RINL antibody.</p