JLP Associates with Kinesin Light Chain 1 through a Novel Leucine Zipper-like Domain

Abstract

Scaffolding proteins exist in eukaryotes to properly assemble signaling proteins into specific multimeric functional complexes. JLP is a novel leucine zipper protein belonging to a family of scaffolding proteins that assemble JNK signaling modules. JLP is a proline-rich protein that contains two leucine zipper domains and a highly conserved C-terminal domain. We have identified kinesin light chain 1 (KLC1) as a binding partner for the second leucine zipper domain of JLP using yeast twohybrid screening. The interaction domain of KLC1 was mapped to its tetratripeptide repeat, which contains a novel leucine zipper-like domain that is crucial for the interaction with JLP. Mutations of Leu-280, Leu-287, Val-294, and Leu-301 within this domain of KLC1 disrupted its ability to associate with JLP. Immunofluorescence studies showed that JLP and KLC1 co-localized in the cytoplasm and that the localization of JLP was dependent on its second leucine zipper. Ectopic expression of a dominant negative form of KLC1 resulted in the mislocalization of endogenous JLP. Moreover, the association between JLP and KLC1 occurred in vivo and was important in the formation of ternary complex with JNK1. These results identify a novel protein-protein interaction between KLC1 and JLP that involves leucine zipper-like domains and support the role of motor proteins in the spatial regulation of signaling modules.

Kinesin Adapter JLP Links PIKfyve to Microtubule-based Endosome-to-Trans-Golgi Network Traffic of Furin

“…Confocal microscopy was then performed in cells expressing relatively low levels of both proteins, because such conditions might more faithfully resemble the localization pattern of the endogenous proteins as observed for PIKfyve (12,43). Consistent with previous studies (37,38,41), in the confocal images we observed that overexpressed JLP L WT attained primarily cytoplasmic and perinuclear distribution. Intriguingly, closer inspection of the deconvoluted confocal images from individual sections taken through the middle of the cell revealed that the JLP L -positive perinuclear structures displayed tubular profiles (Fig.…”

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration

“…We conducted in vitro rescue experiments with both wildtype and altered forms of JSAP1, including a mutant that has a substitution in the leucine zipper domain and cannot interact with KLC, 13 and a deletion mutant lacking the KHC-binding domain (KBD). 26 We used lentiviral vectors to express these wild-type and mutant JSAP1 forms as hemagglutinin (HA)-tagged proteins (designated HA-JSAP1-WT, -LZ and -ΔKBD) in primary hippocampal neurons.…”

JNK-associated Leucine Zipper Protein Functions as a Docking Platform for Polo-like Kinase 1 and Regulation of the Associating Transcription Factor Forkhead Box Protein K1

“…Plasmids-The expression vectors encoding murine S-tagged full-length JLP, JLP domains, and shJLP have been described previously (3,7). Expression vectors for FLAG-hJLP were constructed by subcloning PCR fragments into the EcoRI/ XhoI sites of the pCMV-Tag2B (Agilent Technologies) vector.…”

“…Of all the members characterized in the JIP family, JLP is characterized by the presence of two leucine zipper domains and a JNK binding domain in its N terminus and a conserved C-terminal domain, all of which functions in binding to and forming specific cell signaling complexes (3). Although JLP is ubiquitously expressed, previous studies have shown that the protein plays a role in regulating neurite outgrowth by interacting with SCG10 (4), activating JNK/p38 MAPK signaling (3), regulating cell migration by interacting with G␣12 and G␣13 (5,6), and functioning in vesicle transport by binding to kinesin light chain (KLC1) and dynein microtubule motor proteins (7). Recent studies have identified that JLP and JIP3 can interact with ADP-ribosylation factor 6 (ARF6), a small G protein that function in the regulating cytokinesis by transporting recycling endosomes to the cleavage furrow (8).…”