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

Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, Rapoport TA, Margolis B - J. Cell Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. kverhey@hms.harvard.edu

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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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.
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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.

Mentions: We first looked for an association of kinesin with JIP-1 and with a neuronal-specific upstream kinase of the JNK signal transduction pathway, the DLK (Fan et al. 1996). As seen in Fig. 7 A, immunoprecipitation of kinesin resulted in the coprecipitation of JIP-1 and DLK. A complex of these proteins was also detected in the presence of the detergents digitonin (Fig. 7 A), Triton X-100, or octyl glucoside (data not shown). As seen in Fig. 7 B, precipitation of kinesins by binding to MTs in the presence of the nonhydrolyzable ATP analogue AMP-PNP also resulted in the coprecipitation of JIP-1 and DLK. Importantly, the upstream kinase MKK3 of another MAP kinase pathway, the p38 pathway, was not coprecipitated (Fig. 7 A).


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

Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, Rapoport TA, Margolis B - J. Cell Biol. (2001)

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.
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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.
Mentions: We first looked for an association of kinesin with JIP-1 and with a neuronal-specific upstream kinase of the JNK signal transduction pathway, the DLK (Fan et al. 1996). As seen in Fig. 7 A, immunoprecipitation of kinesin resulted in the coprecipitation of JIP-1 and DLK. A complex of these proteins was also detected in the presence of the detergents digitonin (Fig. 7 A), Triton X-100, or octyl glucoside (data not shown). As seen in Fig. 7 B, precipitation of kinesins by binding to MTs in the presence of the nonhydrolyzable ATP analogue AMP-PNP also resulted in the coprecipitation of JIP-1 and DLK. Importantly, the upstream kinase MKK3 of another MAP kinase pathway, the p38 pathway, was not coprecipitated (Fig. 7 A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. kverhey@hms.harvard.edu

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.

Show MeSH
Related in: MedlinePlus