EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain–binding protein 1–like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1–dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

Effect of DNA-PK dependent phosphorylation of topo-I-S10 on its rate of proteasomal degradation and CPT response.

606 Background: Topoisomerase I (topo-I) degradation by ubiquitin proteasomal pathway (UPP), in response to camptothecins (CPTs) is linked to the CPT response. But the mechanism is not understood. Methods: Improvised GST pull down experiments were performed to isolate topo-I interacting proteins which were analyzed by SDS-PAGE and identified by mass spectrometry. DNA-PKcs mediated phosphorylation of GST-topo-I was analyzed by SDS-PAGE and autoradiography. Immunoblot analysis of anti-BRCA1 precipitates from HCT15 cell lines was performed. GST-topo-I phosphorylated by DNA-PKcs, was incubated with the BRCA1/BARD1 heterodimer in the presence of E1, UbCH5c and ATP in ubiquitination buffer. Four triple negative breast cancer cell lines were selected to compare the degree of topo-I degradation. Colorectal cancer (CRC) tissues were immunostained with 1C1.H5.H7 to determine topo-I-pS10 level and identify a correlation between the topo-I-pS10 level and CPT response. Results: We isolated a topo-I interacting protein complex and determined that Ku70/Ku80/DNA-PKcs complex associates with topo-I, and DNA-PKcs phosphorylates topo-I at Serine10. We showed that cells with higher topo-I-pS10 level rapidly degrade topo-I and are CPT resistant. The cells with non-detectable level of topo-I-pS10 fail to degrade topo-I and are CPT sensitive. Retrospective study with 48 CRC tissues immuno-stained with anti-topo-I-pS10 supported our cell line data. Percent DAB positive nuclei demonstrated statistically significant differential staining between CPT responders and non-responders, and ROC analysis confirmed that nearly all non-responders exhibit >35% positive nuclei, indicating that topo-I-pS10 level determines topo-I degradation and a potential predictive biomarker for CPT response. Conclusions: Colorectal cancers are treated with topo-I inhibitors, but the response rate is low. We have shown that topo-I degradation rate is linked to CPT response. We have determined that high topo-I-pS10 level is indicative of rapid topoI degradation and can serve as a biomarker for CPT resistance.