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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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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.
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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.

Mentions: Given the two-hybrid interaction between KLC and the JIPs and the requisite transport of the JIPs to the plus ends of MTs, we considered the possibility that the JIPs are a cargo of kinesin. We confirmed the interaction between KLC and the JIP proteins by coimmunoprecipitation of HA-tagged KLC and Flag-tagged JIPs expressed together in COS cells. Using anti-Flag antibodies, precipitation of JIP-1, JIP-2, or JIP-3 resulted in coprecipitation of HA-tagged full-length KLC (Fig. 2, lanes 2, 5, and 8). Conversely, anti-HA antibodies precipitated KLC and coprecipitated the Flag-tagged JIP-1, JIP-2, and JIP-3 proteins (Fig. 2, lanes 3, 6, and 9). Identical results were obtained using the HA-tagged TPR motifs of KLC instead of the full-length protein (data not shown). These data confirm a direct interaction between the TPR motifs of KLC and the JIP proteins.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198804&req=5

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.
Mentions: Given the two-hybrid interaction between KLC and the JIPs and the requisite transport of the JIPs to the plus ends of MTs, we considered the possibility that the JIPs are a cargo of kinesin. We confirmed the interaction between KLC and the JIP proteins by coimmunoprecipitation of HA-tagged KLC and Flag-tagged JIPs expressed together in COS cells. Using anti-Flag antibodies, precipitation of JIP-1, JIP-2, or JIP-3 resulted in coprecipitation of HA-tagged full-length KLC (Fig. 2, lanes 2, 5, and 8). Conversely, anti-HA antibodies precipitated KLC and coprecipitated the Flag-tagged JIP-1, JIP-2, and JIP-3 proteins (Fig. 2, lanes 3, 6, and 9). Identical results were obtained using the HA-tagged TPR motifs of KLC instead of the full-length protein (data not shown). These data confirm a direct interaction between the TPR motifs of KLC and the JIP proteins.

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