A novel TIP30 complex regulates endocytic trafficking of signaling receptors by facilitating the transport of vaculolar (H+)-ATPases to early endosomes

TIP30, a 30-kDa HIV-1 Tat-interacting protein, is a tumor suppressor whose expression is altered in a variety of human cancers. Tip30-deficient mice spontaneously develop hepatocellular carcinomas and other tumors as well as mammary preneoplasic lesions. However, the molecular mechanism of TIP30 function remains largely unraveled.;In this study, we show that a novel protein complex consisting of TIP30, Endophilin B1 (Endo B1), acyl-CoA synthetase long-chain family member 4 (ACSL4) interacts with Rab5a, and facilitates Rab5a recruitment to early endosomes by promoting efficient fusion between Rab5a vesicles and endocytic vesicles. Rab5a vesicles are EEA1-negative vesicles carrying vacuolar (H+)-ATPases (V-ATPases), an endosome acidification enzyme that causes ligand-receptor dissociation. Inhibition of TIP30, ACSL4 or Endo B1 impairs Rab5a vesicles loading on early endosomes and causes the mislocalization of V-ATPases, leading to delayed EGF-EGFR dissociation and prolonged EGFR signaling. Furthermore, we show that both arachidonic acid and coenzyme A are essential for the fusion of Rab5a vesicles with endocytic vesicles in vitro. TIP30, ACSL4 and Endo B1can promote vesicle fusion in the presence of arachidonic acid and coenzyme A and can transfer the arachidonyl group to endosomal phosphatidic acid to produce triacylglycerol, which induces membrane tethering and stacking. Together, these results identify a novel function for Rab5a in endocytic trafficking and suggest a mechanism, in which addition of the hydrophobic arachidonyl group to phosphatidic acid by the TIP30 protein complex may bring membranes into close contact to allow for membrane fusion.;Supporting the above findings is the observation that Tip30 deletion dramatically accelerated the onset of mammary tumors in the MMTV-Neu transgenic mouse model of breast cancer, which overexpresses another EGFR family member, HER2/Neu. Similar to liver cells, deletion of Tip30 in mouse mammary cells also caused the trapping of EGF-EGFR complexes in early endosomes, thereby leading to delayed EGFR destruction and sustained EGFR signaling in response to EGF treatment.;We further found that unlike tumors developing in MMTV-Neu mice, almost all of which are estrogen receptor-negative and progesterone receptor-negative (ER-/PR-), tumors arising in Tip30-/-/MMTV-Neu mice are almost exclusively ER+/PR- mammary tumors. Immunofluorescence studies showed that Tip30 is predominantly expressed in ER+ mammary epithelial cells (MECs) and its deletion leads to an increase in the number of phospho-ER positive cells in mammary glands and accelerated activation of Akt in MMTV-Neu mice.