Kinesin adapter JLP links PIKfyve to microtubule-based endosome-to-trans-Golgi network traffic of furin.
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.
Mesh Terms:
1-Phosphatidylinositol 3-Kinase, Adaptor Proteins, Signal Transducing, Alternative Splicing, Animals, Base Sequence, CHO Cells, Consensus Sequence, Cricetinae, Cricetulus, Endosomes, Furin, Golgi Apparatus, Humans, Kinesin, Mice, Microtubules, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary, Protein Transport, Recombinant Fusion Proteins, Two-Hybrid System Techniques
1-Phosphatidylinositol 3-Kinase, Adaptor Proteins, Signal Transducing, Alternative Splicing, Animals, Base Sequence, CHO Cells, Consensus Sequence, Cricetinae, Cricetulus, Endosomes, Furin, Golgi Apparatus, Humans, Kinesin, Mice, Microtubules, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary, Protein Transport, Recombinant Fusion Proteins, Two-Hybrid System Techniques
J. Biol. Chem.
Date: Feb. 06, 2009
PubMed ID: 19056739
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