Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules - PubMed (original) (raw)

Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules

K J Verhey et al. J Cell Biol. 2001.

Abstract

The cargo that the molecular motor kinesin moves along microtubules has been elusive. We searched for binding partners of the COOH terminus of kinesin light chain, which contains tetratricopeptide repeat (TPR) motifs. Three proteins were found, the c-jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) JIP-1, JIP-2, and JIP-3, which are scaffolding proteins for the JNK signaling pathway. Concentration of JIPs in nerve terminals requires kinesin, as evident from the analysis of JIP COOH-terminal mutants and dominant negative kinesin constructs. Coprecipitation experiments suggest that kinesin carries the JIP scaffolds preloaded with cytoplasmic (dual leucine zipper-bearing kinase) and transmembrane signaling molecules (the Reelin receptor, ApoER2). These results demonstrate a direct interaction between conventional kinesin and a cargo, indicate that motor proteins are linked to their membranous cargo via scaffolding proteins, and support a role for motor proteins in spatial regulation of signal transduction pathways.

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Figures

Figure 1

Figure 1

Schematic of protein constructs used in this study. (A) Schematic illustration of the domain structure of KHC, KLC, and their deletion mutants. (B) Schematic illustration of the domain structure of the JIP proteins. The independent overlapping clones isolated in the two-hybrid screen are indicated in red under each protein. JBD, JNK binding domain; coil, predicated coiled coil; L, leucine zipper; SH3, src homology domain 3; PTB, phosphotyrosine binding domain.

Figure 4

Figure 4

The COOH-terminal residues of JIP-1 are required for proper subcellular localization. NIE 115 cells were transiently transfected with the parental plasmid (control) or with plasmids encoding the indicated JIP-1 variants, differentiated, and the expressed proteins were detected by indirect immunofluorescence microscopy using an anti-Myc mAb. Nonspecific background staining is visible in the control cells and is enhanced in A′–F′ to aid in visualization of the cells. Myc–JIP-1 variants were scored as positive for correct cellular localization (JIP-1, JIP-1 [307–711], and JIP-1 [P704A]) if fluorescence was more pronounced at the neurite tips (arrowheads), whereas transfected proteins were considered negative for localization (JIP-1 [307–700] and JIP-1 [Y709A]) if fluorescence was observed to be more prominent in the cell body (arrows).

Figure 2

Figure 2

Coimmunoprecipitation of KLC and the JIP proteins. Lysates of COS cells expressing Flag-tagged JIP-1, JIP-2, or JIP-3 together with HA-tagged KLC were immunoprecipitated (IP) with no primary antibody (−), with an anti-Flag mAb (F), or with an anti-HA mAb (H). Precipitates were immunoblotted to detect the expressed proteins using polyclonal antibodies to both epitope tags.

Figure 3

Figure 3

The COOH-terminal residues of JIP-1 are required for interaction with the KLC TPRs. (A) Sequence alignment of the COOH-terminal residues of JIP-1 and JIP-2 across species. The 11 residues deleted in the mutant JIP-1 (307–700) are indicated (boxed and shaded) as are the residues mutated in JIP-1 (P704A) and JIP-1 (Y709A) (*). hJIP-1/IB1, human JIP-1; mJIP-1b, mouse JIP-1b; hJIP-2, human JIP-2; pJIP-2, pig JIP-2 derived from a partial EST (accession AW312953); dJIP/SP512, Drosophila JIP-1/2; ceJIP, C. elegans JIP sequence derived from cosmid C13A10. (B) Schematic illustration of the Myc-tagged JIP-1 constructs used in this study. (C) Lysates of 293 cells expressing Myc-tagged JIP-1 (307–711), JIP-1 (307–700), JIP-1 (P704A), or JIP-1 (Y709A) together with HA-tagged KLC TPRs were immunoprecipitated (IP) for the KLC TPRs using an anti-HA mAb (top) or for the JIP-1 variants using an anti-Myc mAb (middle). Precipitates were immunoblotted (IB) for associated proteins using anti-Myc (top) or anti-HA polyclonal antibodies (middle). The ∼65-kD band in the top panel is the heavy chain of immunoglobulin. Lysates were immunoblotted to detect the expressed proteins (bottom).

Figure 5

Figure 5

Expression of kinesin dominant negative constructs causes mislocalization of endogenous JIP-1 protein. CAD cells were transiently transfected with plasmids encoding the HA-tagged KLC TPRs (A), HA-tagged PP5 TPRs (B), HA-tagged KLC truncation KLC-176 (C), or Myc-tagged KHC truncation KHC-891 (D). After differentiation, the expressed proteins were detected by indirect immunofluorescence microscopy using mAbs to the epitope tags (left). Endogenous JIP-1 protein was detected with an affinity-purified polyclonal antibody (right). Note that the background fluorescence has been enhanced to show the entire neuronal cell. Arrowheads denote tips of transfected cells; arrows denote tips of untransfected cells.

Figure 6

Figure 6

A kinesin dominant negative construct causes mislocalization of expressed JIP-2 and JIP-3 proteins but not endogenous mitochondria, neurofilaments, or MTs. (A and B) CAD cells were transiently transfected with plasmids encoding the HA-tagged KLC TPRs together with Flag-tagged JIP-2 (A) or Flag-tagged JIP-3 (B). After differentiation, the expressed proteins were detected by indirect immunofluorescence microscopy using a mAb to the HA tag (left) and a polyclonal antibody to the Flag tag (right). Arrowheads denote tips of transfected cells; arrows denote tips of untransfected cells. (C–E) CAD cells were transiently transfected with a plasmid encoding the HA-tagged KLC TPRs. After differentiation, the expressed protein was detected by indirect immunofluorescence microscopy using a polyclonal antibody to the HA tag (left). Endogenous MTs, neurofilaments (NF), and mitochondria were detected with mAbs to tubulin (C, right) or neurofilaments H + M (D, right), or with MitoTracker (E, right). Note that in C, the cells were only allowed to differentiate for 12 h, so that MT organization could be assessed before the cell bodies rounded up.

Figure 7

Figure 7

Kinesin and JIP-1 are associated with the upstream kinase DLK and the transmembrane receptor ApoER2. (A) An mAb to KHC (H2) was used to immunoprecipitate (IP) kinesin and associated proteins from a rat brain high speed supernatant in the presence of digitonin or no detergent. The presence of associated polypeptides was detected by immunoblotting the precipitates with polyclonal antibodies to the indicated proteins. (B) Rat brain high speed supernatant was subjected to an MT binding assay in the presence of Triton X-100 by adding ATP, AMP-PNP, and/or MTs as indicated. MTs and bound proteins were sedimented through a sucrose cushion, and the presence of the indicated proteins in the MT pellets was detected by immunoblotting. (C) CAD cells were transiently transfected with a plasmid encoding the HA-tagged KLC TPRs. After differentiation, the expressed protein was detected by indirect immunofluorescence microscopy using a mAb to the HA tag (left). Endogenous DLK kinase was detected with a polyclonal antibody (right). Arrowheads denote tips of transfected cells; arrows denote the tip of an untransfected cell.

Figure 8

Figure 8

Model for the transport of cargo by kinesins. JIP proteins form a scaffold, on which cytoplasmic as well as plasma membrane proteins are assembled. The entire complex is transported down an axonal process by conventional kinesin. Note that the JIP proteins are known to form homodimers and heterodimers, although only one polypeptide is drawn for clarity. Similarly, LIN-2, -7, and -10 form a scaffold, on which cytoplasmic and transmembrane proteins assemble. The entire complex is transported down a dendritic process by the kinesin superfamily member KIF17.

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