title: Kinesin adapter JLP links PIKfyve to microtubule-based endosome-to-trans-Golgi network traffic of furin.
abstract: JIPs (c-Jun N-terminal kinase interacting proteins), which scaffold JNK/p38 MAP kinase signaling modules, also bind conventional kinesins and are implicated in microtubule-based membrane trafficking in neuronal cells. Here we have identified a novel splice variant of the Jip4 gene product JLP(L) (JNK-interacting leucine zipper protein) in yeast-two hybrid screens with the phosphoinositide kinase PIKfyve. The interaction was confirmed by pulldown and coimmunoprecipitation assays in native cells. It engages the PIKfyve cpn60_TCP1 consensus sequence and the last 75 residues of the JLP C terminus. Subpopulations of both proteins cofractionated and populated similar structures at the cell perinuclear region. Because PIKfyve is essential in endosome-to-trans-Golgi network (TGN) cargo transport, we tested whether JLP is a PIKfyve functional partner in this trafficking pathway. Short interfering RNA (siRNA)-mediated depletion of endogenous JLP or PIKfyve profoundly delayed the microtubule-based transport of chimeric furin (Tac-furin) from endosomes to the TGN in a CHO cell line, which was rescued upon ectopic expression of siRNA-resistant JLP or PIKfyve constructs. Peptides from the contact sites in PIKfyve and JLP, or a dominant-negative PIKfyve mutant introduced into cells by ectopic expression or microinjection, induced a similar defect. Because Tac-TGN38 delivery from endosomes to the TGN, unlike that of Tac-furin, does not require intact microtubules, we monitored the effect of JLP and PIKfyve depletion or the interacting peptides administration on Tac-TGN38 trafficking. Remarkably, neither maneuver altered the Tac-TGN38 delivery to the TGN. Our data indicate that JLP interacts with PIKfyve and that both proteins and their association are required in microtubule-based, but not in microtubule-independent, endosome-to-TGN cargo transport.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Ognian C
authors.initials: OC
authors.lastname: Ikonomov
authors.forename: Jason
authors.initials: J
authors.lastname: Fligger
authors.forename: Diego
authors.initials: D
authors.lastname: Sbrissa
authors.forename: Rajeswari
authors.initials: R
authors.lastname: Dondapati
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Mlak
authors.forename: Robert
authors.initials: R
authors.lastname: Deeb
authors.forename: Assia
authors.initials: A
authors.lastname: Shisheva
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D016384:Consensus Sequence / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D045683:Furin / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D045683:Furin; D016547:Kinesins; D019869:Phosphatidylinositol 3-Kinases; D011993:Recombinant Fusion Proteins; C468928:Spag9 protein, mouse; C492139:PIKFYVE protein, human; C116930:Pikfyve protein, mouse; C468731:FURIN protein, human